Monday, February 20, 2012

Gram staining - Procedure, mechanism, explanation

INTRODUCTION The Gram stain was developed in 1884 by the
Danish bacteriologist Hans Christian Gram. It is one of the most useful
staining procedures because it classifies bacteria into two large groups: 1) gram-positive
and 2) gram-negative.

PROCEDURE

1) A heat-fixed smear is flooded with a basic purple dye, usually
crystal violet. Because the purple stain imparts its color to all cells, it is
referred to as a primary stain.

2) After 1 minute, the crystal violet is drained off and washed with distilled water. The smear
is then covered with Gram's iodine, a mordant or helper. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.

3) Next, the slide is
washed with alcohol (95% ethanol) or an alcohol-acetone solution. This solution is a
decolorizing agent which removes the purple from the cells of some species but
not from others. When the procedure is carried out, the slide is held at an angle and 95% ethanol is poured until the draining solution no longer has a purple tint.

4) The alcohol is now rinsed
off with distilled water and the slide is then stained with safranin, a basic red dye known as the counter stain. The staining process is allowed to be carried out for 2-3 minutes. The smear is
washed again, heat dried and examined microscopically.

MECHANISM

The purple dye and the iodine combine in the cytoplasm of each
bacterium and color it dark violet or purple. Bacteria that retain this color
after the alcohol has attempted to decolorize them are classified as
gram-positive. Bacteria that lose the dark violet or purple color after decolorization
are classified as gram negative.

Because gram-negative bacteria are colorless
after the alcohol wash, they are no longer visible. This is why the basic dye
safranin is applied. It turns the gram-negative bacteria pink. Stains such as
safranin that have a contrasting color to the primary stain are called counter stains.
Because gram-positive bacteria retain the original purple stain, they are not
affected by the safranin counter stain.

EXPLANATION

Gram-positive bacteria have a thicker peptidoglycan
(disaccharides and amino acids) cell wall than gram-negative bacteria. The latter contain a layer of lipopolysaccharide (lipids
and polysaccharides) as part of their cell wall.

When applied to both
gram-positive and gram-negative cells, crystal violet and then iodine readily
enter the cells. Inside the cells, the crystal violet and iodine combine to
form the crystal violet-iodine (CV- I) complex. This complex is larger than the crystal violet molecule that
entered the cells and because of its size, it cannot be washed out of the
intact peptidoglycan layer of gram-positive cells by alcohol. Consequently,
gram-positive cells retain the color of the crystal violet dye.

In gram-negative
cells, however, the alcohol wash disrupts the outer lipopolysaccharide layer and the CV- I complex is washed out through the thin layer of peptidoglycan .
As a result, gram negative cells are colorless until counterstained with
safranin, after which they are pink.

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